Attenuation as a control in gene expression.
Attenuation as a control in gene expression.
Attenuation in the tryptophan biosynthetic operon of E. coli.
- References
- Yanofsky, Nature 289:751 (1981) Read
- Genes VI pp374-380
Attenuation was first observed by Charles Yanofsky in the trp operon of E. coli. The first observation was linked to two separate scientific facts. Mutations which knocked out the trp R (repressor) gene still showed some regulation of the trp operon (these mutants were not fully induced/repressed by tryptophan). The total range of trp operon regulation is about 700 X (on/off). When the trp repressor was knocked out, one still got about 10 X regulation by the absence or presence of trp. When the sequence of the beginning of the trp operon was determined an unusual open reading frame (ORF) was seen immediately preceding the ORFs for the known structural genes for the tryptophan biosynthetic enzymes.
The general structural information shown below was observed from the sequence of the trp operon.
First, Yanofsky observed that the ORF contained two tandem Trp codons and the protein had a Trp percent composition which was about 10X normal. Second, the mRNA in this region contained regions of dyad symmetry which would allow it to form two mutually exclusive secondary structures. One of the structures looked exactly like a rho-independent transcription termination signal. The other secondary structure, if formed, would prevent the formation of this secondary structure and thus the terminator. This other structure is called the "preemptor".
Mechanism of attenuation in the trp operon.
The proposed mechanism of how this mRNA secondary structure and the trp leader peptide could regulated transcription of the trp biosynthetic enzymes includes the following.
- RNAP initiates transcription of the trp promoter.
- RNAP pauses at about nucleotide 90 at a secondary structure (?the first one shown above?).
- Ribosomes engage this nascent mRNA and initiate
translation of the leader peptide.
- RNAP is then "released" from its pause and continues transcription.
- When RNAP reaches the region of the potential terminator,
whether it continues or not is dependent on the position
of the ribosome "trailing behind".
- If the ribosome, stalls at the tandem Trp codons, waiting for the appropriate tRNA, region 1 is sequestered within the ribosome and thus cannot base pair with region 2. This means that region 2 and 3 become based paired before region 4 can be transcribed. This forces region 4 when it is made to be single stranded, preventing the formation of the region 3/4 terminator structure. Transcription will then continue.
- If the ribosome translates the leader peptide with no hesitation, it then covers a portion of region 2 preventing it from base pairing with region 3. Then when region 4 is transcribed, it forms a stem and loop with region 3 and transcription is terminated, generating a ca. 140 base transcript.
- This mechanism of control measures the amount of available, charged Trp-tRNA.
The location of ribosomes determines which alternate secondary structures form.
Other operons controlled by attenuation.
The discovery of this type of mechanism to control the expression of genes in a biosynthetic operon lead to its rediscovery in a wide variety of such operons for which repressors had never been discovered. For example:
| Operon | Leader peptide |
|---|---|
| Histidine | MTRVQFKHHHHHHHPD stop |
| Threonine | MKRISTTITTTITITTGNGAG stop |
| Ilv (GEDA) | MTALLRVISLVVISVVVIIIPPCGAALGRGKA stop |
| Leu | MSHIVRFTGLLLLNAFIVRGRPVGGIQH stop |
| Phenylalanine | MKHIPFFFAFFFTFP stop |
| IlvB | MTTSMLNAKLLPTAPSAAVVVVRVVVVVGNAP stop |
| Ala = A (8.6) | Arg = R (4.9) |
| Gly = G (8.4) | Ile = I (4.5) |
| Leu = L (7.4) | Asn = N (4.3) |
| Ser = S (7.0) | Gln = Q (3.9) |
| Val = V (6.6) | Phe = F (3.6) |
| Lys = K (6.6) | Tyr = Y (3.4) |
| Thr = T (6.1) | Cys = C (2.9) |
| Glu = E (6.0) | His = H (2.0) |
| Asp = D (5.5) | Met = M (1.7) |
| Pro = P (5.2) | Trp = W (1.3) |
An Outside Link to Discussion of Attenuation
Attenuation control of b-lactamase gene expression.
Jaurin et al., Nature 290:221-225 (1981)
Attenuation also controls operons other than biosynthetic ones. One such example is the control of chromosomal b-lactamase in E. coli. This enzyme is regulated by growth rate, the faster the organism grows, the higher the cellular concentration of b-lactamase. The leader RNA sequence upstream of the structural gene for b-lactamase has the following sequence:
The suggested mechanism for how this sequence/structure regulates the rate of expression of b-lactamase which starts at the far right AUG (double underline) goes as follows:
- E. coli contain (per cell) much higher concentrations of ribosomes at high growth rates. As a matter of fact at low growth rates ribosomes are limiting.
- As the growth rate of E. coli increases, the single underlined start codon of the above sequence becomes occupied more often with ribosomes which are attempting to initiate protein synthesis at this AUG codon and immediately terminating at the adjacent UAA codon.
- The proposed stem and loop structure immediately upstream from the boxed UUUU sequence cannot form due to the presence of ribosomes.
- This disrupts this potential r-independent termination sequence and transcription can continue past the UUUU and the b-lactamase gene is transcribed.
- This mechanism is measuring the cellular concentration of ribosomes capable of initiating translation which is directly proportional to growth rate.
Here's what happens to cells when they are exposed to penicillin.
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